Methods and compositions of a hybrid genetic corn complement, DK743

ABSTRACT

This invention relates generally to the production of maize, commonly known in the United States as corn, and more specifically to hybrid corn plants with certain advantageous phenotypes resulting from interactions of the haploid genetic contributions of inbred parental lines. This invention relates to the hybrid genetic complement, the expression of which produces these phenotypes and to the complement as housed in seeds and tissues, in particular, those capable of producing or regenerating the hybrid plants either in vivo or in vitro. An aspect of this invention, hybrid DK743, is characterized by many advantageous phenotypic traits including superior yield and staygreen. It has characteristic restriction fragment length polymorphism (RFLP) and isozyme profiles.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the production of maize, and morespecifically to hybrid corn plants with certain advantageous phenotypesresulting from interactions of the haploid genetic contributions ofinbred parental lines. Seeds and tissues, in particular, those capableof producing or regenerating the hybrid plants either in vivo or invitro are disclosed. An aspect of this invention, hybrid DK743, ischaracterized by many advantageous phenotypic traits including excellentgrain yield and silage production, staygreen and disease tolerance.Although these characteristics are particularly suited for southernclimates, an advantage of this genetic complement is good performance ofplants expressed over a wide geographical area. DK743 has characteristicrestriction fragment length polymorphism (RFLP) and isozyme profiles.

2. Description of the Related Art

Crop improvement has been a major focus of human agriculturists sincethe hunting gathering societies moved into the agricultural phase ofhuman existence. Early crude attempts to improve crops focused on thechoice of parental plants to become the progenitors of the nextgeneration, a choice made on the readily detectable characteristics ofthe parents. The objective was to produce offspring having theadvantageous traits of the parents. However, from what we now know ofgenetics and genetic theory, such efforts were usually doomed tofailure--in some instances because the parental phenotypes could not bereconstructed in their offspring due to disruption of the geneticcomplements of the parents by segregation of a diploid complement intohaploid gametes, and shuffling of the genetic material by recombination.Even worse, certain combinations of parental genomes yielded deleteriouseffects due to interactions of genes at the same or different loci. As aconsequence, success at crop improvement was painstakingly slow,sporadic and rarely reproducible.

Modern sophisticated crop breeding of the 1900's has benefitted fromknowledge gained by Gregor Mendel, which was published in 1865 andrediscovered in the early 1900's. That work and subsequent work ofothers indicated that both single gene (mendelian) and polygenic controlmust be considered when planning breeding programs to improve cropcharacteristics. In fact, all corn as we know it today, Zea mays, is aresult of human manipulation. It is not a plant occurring sporadicallyin nature.

Despite much knowledge that has developed subsequently, each breedingprogram represents at least in part a new attempt to mold the plantgermplasm into new and more productive, more desirable phenotypes. Thismolding process benefits from the development over many years of inbredlines. These lines are not found in the wild, that is, in naturalsettings, and by themselves are generally not commercially productive.However, inbred lines are valuable as repositories for genes that arepreserved in relatively stable conditions due to the true-breedingcapabilities of these genetically uniform lines. Such genes are thenavailable to be repeatedly tested for their effects in various breedingcombinations and to be incorporated into commercially desirable crops.

Inbred lines are those that are essentially homozygous. Initial repeatedinbreeding concentrates a subset of ancestral genes in offspring. Linesmay also be improved in this fashion. The more genetically similar theancestral lines, the more rapid the approach to homozygosity.Self-fertilization is a way of maintaining homozygosity. Homozygosityrefers to the condition of the genetic complement in which the paireddiploid positions at each locus are occupied by identical alleles.Alleles are conditions of a gene which differ in their nucleotidesequences. Alleles are recognizable when they are expressed as distinctphenotypes. Homozygosity in an inbred line is achieved by repeatedinbreeding. In general, by the sixth or seventh generation, the inbredline is considered genetically pure, or "true-breeding" althoughspontaneous changes in the genetic material (mutations) and other eventsmay preclude absolute homozygosity. Environmental variations may producesome phenotypic variability even within genetically identically lines.

Unfortunately, reduction in yield performance and the appearance ofother plant characteristics which are undesirable accompaniesinbreeding. In addition, progressive selfing (self-fertilization) ofinbred lines reduces plant vigor. Many of these deleterious effects arecaused by homozygosity for deleterious recessive genes whose effects areunmasked by loss of desirable dominant alleles. Consequently, inbredcorn lines per se are not grown to be used as commercial crops. However,they are extremely important as vehicles to preserve genes and toproduce first generation (F₁) hybrids by the process of hybridization bycross-breeding.

Hybrid plants are likely to be heterozygous at many loci, as opposed tobeing homozygous, like the inbred parental lines. Heterozygosity refersto the fact that at a locus, there are different conditions of a gene(different alleles). One desirable result of crossing two inbred linesis that hybrid vigor or heterosis may arise wherein the hybrid plantsproduced have markedly improved higher yields, better stalks, betterroots, better uniformity and better insect and disease resistance, thaneither inbred parent. For corn used as animal feed, one of the goals isdecreasing the amount of feed needed for animal weight gain.

Furthermore, as result of self-pollination of these F_(l) hybrid plants,a process possible in plants such as corn which have both male andfemale sex organs on the same plant or of cross-pollination of F₁ hybridplants, a second generation (S₁ or F₂) hybrid may be produced.Non-parental genetic combinations occur in these offspring due toindependent assortment at meiosis of genes on different chromosomes andby recombination of genes on homologous (matched and paired at meiosis)chromosomes. Because of this further shuffling of the genetic materialfrom the F₁ into the F₂, some of the F₂ hybrid plants produce lessdesirable plants than those of the F₁ in terms of the traits discussedabove, due to homozygosity for deleterious recessive alleles and otherdisruption of the F₁ genetic complement. In addition, there is increasedvariability overall of trait performance in the F₂ due to this extensivegenetic shuffling, in particular, if many loci are involved incontrolling a particular trait. It is not generally beneficial,therefore, for farmers to save the seed of F₁ hybrids. Rather, a cycleof purchase by farmers each year of F₁ hybrid seed for planting is therule. Seed corn companies attempt to market new improved seed eachseason to attract these consumers.

North American farmers plant over 70 million acres of corn at thepresent time. There are extensive national and international programs incommercial corn breeding. Clearly this endeavor has a major impact onhumanity in the form of food production. Basic methods of cross breedinginbred lines to produce hybrids are known in concept by those skilled inthe art. However, actual manipulation of these basic methods to generateimproved hybrids is a delicate, arduous and sophisticated process.Breeders armed with methods to physically control plant breeding, andwith an array of inbred lines with various known phenotypic traits,cannot expect to merely go into the field with these inbred lines, breedthem using well-established general methods, and walk out of theirlaboratories, greenhouses and fields with superior hybrids.

One of the first difficulties encountered is in breeding superior inbredparental lines, due to the difficulties discussed above which areinherent in inbreeding, for example, reduced vigor.

The skilled corn breeder also must make determinations regarding whichcombinations of these inbred lines should be selected to produceimproved hybrids. The traits selected for commercial desirability aregenerally not expressions of genes operating in a vacuum. Rather, toproduce a plant which as a whole has an array of desirablecharacteristics, there must be a balance in terms of improvements.Phenotypic traits may show positive or negative correlations withininbred lines and between those lines and their hybrid progeny.Consequently, improving one trait may lead to poor outcome of another.Furthermore, hybrid plants that are beneficial in one set ofenvironmental conditions may do poorly in others.

With the desperate need for increased food production within diverseareas of the world, and for growing various crops in different locationsof the world for maximum and control of local persons over theiragricultural destiny, it is important to develop wide ranges of hybridsthat are going to perform well in both specific and general ecologicaland commercial niches.

Evidence of the difficulties inherent in commercial crop breeding isprovided by the continual and highly competitive research in both thelaboratory and the field revolving around improvement of inbred andhybrid lines. Removal of some of the uncertainty in large scale andexpensive field testing is resulting from the application of methods ofmolecular biology whereby segments of the genetic complement may besingled out for faster, more selective and more successful breeding, andgenetic complements may be combined in vitro, that is, in laboratorytissue culture vessels rather than in corn fields.

Some of the phenotypic traits for which improvements have continuallybeen sought by hybridization of corn, include the yield; stalks; roots;resistance to insecticides, pests and disease; and markedly more uniformcharacteristics. With mechanical harvesting of many crops, uniformity ofplant characteristics such as germination and stand establishment,growth rate, maturity, and fruit size, is important. Other desirablephenotypic characteristics for field crops include tolerance to heat anddrought, reduced time to crop maturity, and better agronomic quality.However, despite some successes in breeding programs in the 1900's,progress is painstakingly slow--each qualitative improvementrepresenting a small quantitative step.

Currently, it appears as if there is polygenic control over mostcommercially desirable traits such as yield. This means that many genes,generally on many chromosomes, contribute to the phenotypic appearanceof the plant. The variance of the trait in inbred lines is less thanthat expected in hybrids formed from inbreds because of intralocus andinterlocus interactions. Consequently, selective breeding programs toimprove crops are not completely predictable. The development of inbredlines generally requires at least about 5 to 7 generations of selfing.Inbred lines are then cross-bred in an attempt to develop improved F₁hybrids.

SUMMARY OF THE INVENTION

Corn is the most important crop in the United States (Duvick, 1984).Consequently, a great deal of effort is expended to increase cornproduction and improve quality. A major method to achieve these goals isto produce hybrid plants which are superior to their inbred parents andto other hybrids.

This invention addresses some of the shortcomings in the prior art ofcorn hybridization, and discloses a corn hybrid genetic complement that,when expressed in the form of a corn plant, exhibits superiorcharacteristics that include excellent performance for both grain yieldand silage production, staygreen and disease tolerance. Yield is a traitof major commercial interest because it measures grain output per acre.Staygreen is a measure of general health of the plant. Silage productionrefers to bulk of stored plant material.

"Genetic complement" refers to that aggregate of nucleotide sequencesthat, when expressed in corn cells, yields a phenotype in corn plants,or components of plants including cultured cells, which includesphenotypic traits within specified quality and quantity ranges. As anexample of a superior phenotypic trait of DK743, the improved yield ofthe hybrid of this invention compared to two commercially successfulhybrids of similar maturity is dramatic, from between 3.8 to 7.6 bushelsper acre. This is to be contrasted with the increase in U.S. corn yieldof about 2 bushels per acre in recent times reported by Troyer (1990).This improvement was equated by Troyer to a value of about $330 millionper year for the United States.

A method used to produce the hybrid genetic complement of the presentinvention was to combine the genetic complements of two different inbredlines. These lines were produced by repeated crossing of ancestrallyrelated corn plants to concentrate certain genes within the inbredlines.

More specifically, the hybrid genetic complement of the presentinvention was produced by interbreeding two corn lines which differ intheir allelic constitutions at least at some loci. Alleles areconditions of genes that generally occupy the same locus, or position ona chromosome. Genes are sequences of nucleic acids, more specifically,DNA. Different alleles are characterized by different sequences of theDNA. These sequences are still capable of occurring at particular loci,although because of their sequence variation, they may be transcribed asmRNA in different nucleic acid sequences. Depending on the codingequivalency of the altered sequences, there may be changes in thetranslation into amino acid sequences in the gene products.

In an illustrative embodiment, the parental complements were contributedto the hybrid of this invention by transmission as haploid complements,that is, in the form of gametes, each gamete comprising one member ofthe pair of alleles at each locus. More particularly, this inventionrelates to a hybrid genetic complement formed by the combination of ahaploid genetic complement from each of the inbred lines of corndesignated respectively, F118 and HBA1. F118 is a proprietary line. HBA1is an inbred line which has been patented (U.S. Pat. No. 4,594,810). Thehybrid complement is designated DK743.

Because the parents are members of inbred lines, all haploid complementsderived from the parent are expected to be essentially the samegenetically, with the exception of, for example, mutations and thepresence of heterozygous loci if there was not 100% homozygosity.

Although the genetic complements of the inbred parental lines will begenerally the same as that contained in haploid pollen or eggs, theremay be some effects on offspring of maternal cytoplasmic factors. Inaddition, one of the parental lines may be preferred as the male and theother as the female due to phenotypic characteristics of the parentalplant that affect reproduction. For example, one of the lines may havehigher seed yield, one may shed pollen better, one may have preferredseed or tassel characteristics.

In embodiments wherein the hybrid genetic complement is produced byfield breeding schemes or by another method wherein gametes designatedas pollen and egg are combined, the preferred source for the maternalhaploid complement is F118 and for the paternal haploid complement HBA1.HBA1 is preferred as a paternal rather than maternal source because ofpoor seed quality from this line.

Because the inbred lines by definition are homozygous at most loci, theresulting hybrid is likely to be heterozygous at most loci unless theparental lines had the same allelic complement at some loci, perhaps dueto common ancestry. The goal of the corn breeder for this invention wasto produce heterosis, a phenomenon wherein the heterozygote produces aphenotype that is more desirable than that exhibited by either parent.In the hybrid genetic complement disclosed herein, that goal has beenachieved.

The complement designated DK743 when expressed as a plant or itscomponents thereof, has superior advantages when compared to otherhybrid competitors of similar maturity. Maturity is a concept well knownto those skilled in the art and refers to the observation that plants ofdifferent genotypes take different times to mature. This period must fitthe temperature range of the environment in which the plants are raisedto permit reproduction (FIG. 1). In other words, if a plant does notmature before the temperature drops below a permissible level, the plantcan not be successful.

The hybrid corn plant designated DK743 typically has a relative maturityof about 123 days. It is particularly suited for growth in Southernclimates, e.g. the 120 and 125 RM zones from North Carolina toCalifornia. (See FIG. 1). It typically produces significantly higheryields, 3.8 to 7.6 bushels per acre, based on FACT trials. FACT is anacronym for Field Analysis Comparison Trials (strip trials), on-farmtesting programs to perform final evaluations of the commercialpotential of a product. These performance and comparison trials areperformed on actual farms under conditions which approximate the actualgrowing conditions used by farmers who will eventually purchase theseeds of the hybrid. These are "real life" trials to follow up andsupport research testing. Research testing is a more vigorous evaluationof basic characteristics of hybrids on smaller plots of land.

DK743 is a very widely adapted and versatile 124 RM (see FIG. 1) singlecross corn hybrid that exhibits excellent performance for both grainyield and silage production. DK743 has excellent staygreen and diseasetolerance needed for Southern corn production. In addition to havingexcellent resistance to Southern corn leaf blight, DK743 also shows goodresistance to the Southeastern Virus Complex. DK743 performs well forboth yield and silage all across the 120 and 125 RM zones from NorthCarolina to California.

It is very rare and unique for a corn hybrid to possess the excellentversatile traits of DK743 and also be adapted over such a widegeographical area.

For methods of combining the F118 and HBA1 genetic complements, otherthan breeding in fields or greenhouses, for example, in vitrocombinations performed in tissue culture, there need be no preferredmaternal or paternal designation. Moreover, the genetic complementscombined in vitro may represent only the subset of genes necessary toproduce the superior phenotype of DK743. These nucleic acid segments maybe determined by mapping of quantitative loci using discrete markerse.g., by the QTL method, (See Asins (1988); Beckmann and Soller (1983);Burr et al. (1983); Lande and Thompson, (1990); Lander and Botstein(1989); Paterson et al., (1988); EP 0 306 139 A2; PCT/US89/00709).Nucleic acid segments that have no necessary coding function, need notbe included.

The hybrid genetic complement designated DK743 exists in all somatic(non-germinal) cells of a corn plant, and is claimed in all thoseaspects, in particular, roots, stems, leaves, seeds and all floweringparts, including pollen grains. All these parts of plants derived fromthe hybrid DK743 are claimed because all have the novel geneticcomplement of this invention.

The pollen grains are haploid samples of the diploid genetic complementof the hybrid. These haploid samples result from independent segregationof the individual maize chromosomes and shuffling of the genes onhomologous chromosomes by the natural recurring phenomenon ofrecombination at meiosis.

The hybrid genetic complement may be produced in several ways, either invivo or in vitro. The following in vivo method comprises a breedingscheme which may be used to produce a hybrid corn plant in the field:

(a) planting in pollinating proximity seeds from inbred corn lineshaving the designations, F118 (ATCC Accession #75745) and HBA1 (ATCCAccession #40225)

(b) cultivating corn plants resulting from the planting before the timeof flowering;

(c) emasculating the plants of inbred corn line F118;

(d) allowing cross-pollination to occur between said corn lines; and

(e) harvesting seeds produced by the plants of the emasculated cornplants from the line designated F118.

Hybrid plants may be grown from seeds with the genetic complementdisclosed herein by methods well known to those skilled in the art.These seeds have been deposited with the ATCC as Accession No. 75186.

This invention also relates to the hybrid genetic complement containedin seeds. Plants grown from these seeds by methods well known to thoseskilled in the art are expected to exhibit the characteristics listed inTables 1 through 5.

To produce the hybrid genetic complement in vitro, a plant may beregenerated from cells in culture (Gordon-Kamm, et al., 1990). Toregenerate hybrid plants, cells are obtained which comprise the hybridgenetic complement, for example, somatic cells from a DK743 corn plant.These cells are then cultured in vitro in a media comprising anembryogenic promoting hormone until callus organization is observed. Atthis point, cells are transferred to media which includes a tissueorganization promoting hormone. After tissue organization is observed,the cells are subcultured onto media without said hormone, to allow forshoot elongation or root development. Finally, the plantlets aretransferred onto a minimal medium to provide for hardening of the plant.

Embodiments of the embryogenic promoting medium are dicamba, 2,4-D andthe like. Embodiments of the tissue organization promoting mediumcomprises BAP (6-benzylaminopurine), myoinositol,2,4-dichlorophenoxyacetic acid (2,4-D), ABA (absciscic acid), NAA(naphthol acetic acid), IAA (indole acetic acid) and 2IP(2-iminopurine). IBA may be used to stimulate rooting. Minimal mediacomprises Clark's media.

The combination of genetic complements of the inbred lines F118 and HBA1to yield the hybrid complement disclosed herein is claimed within thescope of this invention regardless of the method used to produce it.

A deposit of 2500 seeds each of inbred plants designated F118 and HBA1was made with the American Type Culture Collection, Rockville PikeBethesda, Md. on Apr. 18, 1994 for F118 and on Mar. 12, 1986 for HBA1.These deposited seeds have been designated, for public accessionpurposes, as 75745 for F118, and 40225 for HBA1. These deposits weremade in accordance with the terms and provisions of the Budapest treatyrelating to deposit of microorganisms.

The deposits are made for a term of at least thirty years and at leastfive years after the most recent request for the furnishing of a sampleof the deposits was received by the depository. Prior to making thosedeposits, Applicants state that seeds of inbred corn plants F118 andHBA1 are on deposit at DeKalb Genetics Corporation, Sycamore, Ill., andthat such seeds are accessible and available to the Commissioner ofPatents and Trademarks during pendency of this application.

Phenotypic traits characteristic of the expression of the hybrid geneticcomplement of this invention include those that are distinguishable byelectrophoretic separation of DNA sequences cleaved by variousrestriction endonucleases. These traits (genetic markers) are termedRFLP (restriction fragment length polymorphisms).

Restriction fragment length polymorphisms (RFLPs) are geneticdifferences detectable by DNA fragment lengths, typically revealed byagarose gel electrophoresis, after restriction endonuclease digestion ofDNA. There are large numbers of restriction endonucleases available,characterized by their nucleotide cleavage sites and their source, e.g.,the bacteria E. coli. Variations in RFLP's result from nucleotide basepair differences which alter the cleavage sites of the restrictionendonucleases, yielding different sized fragments.

Restriction fragment length polymorphism analysis was conducted byNative Plants Incorporated (NPI) (Table 3). This service is available tothe public on a contractual basis from Linkage Genetics (Table 4). Forthis analysis, the genetic marker profile of the parental inbred lineswere determined. Because these inbreds are essentially homozygous at allrelevant loci, they should have only one allele at each locus.Consequently, the diploid genetic marker profile of the hybrid offspringof the inbred parents should be the sum of those parents, e.g., if oneparent had the allele A at a particular locus, and the other parent hadB, the hybrid AB is by inference. The RFLP complement is presented inTable 3.

Probes were prepared to the fragment sequences, these probes beingcomplementary to the sequences thereby being capable of hybridizing tothem under appropriate conditions well known to those skilled in theart. These probes are labelled with radioactive isotopes or fluorescentdyes for ease of detection. After the fragments are separated by size,they were identified by the probes. Hybridization with a unique clonedsequence permits the identification of a specific chromosomal region(locus). Because all alleles at a locus are detectable, RFLP's arecodominant alleles, thereby satisfying a criteria for a genetic marker.They differ from some other types of markers, e.g, from isozymes, inthat they reflect the primary DNA sequence, they are not products oftranscription or translation. Furthermore, different RFLP profilesresult from different arrays of restriction endonucleases.

Other characteristic phenotypic traits include the isozyme variantslisted in Table 4. These are codominant genetic markers that delineatesegments of nucleic acids characterizing the genetic complement.

An important use of genetic markers is to reconstruct ("reverseengineer") the parental genetic complements of an offspring. Forexample, the hybrid genetic complement results from the combination oftwo inbred parental complements. The genetic complements of the parentallines may be determined by comparing the RFLP and isozyme geneticprofiles of the hybrid and that of HBA1, a patented line (U.S. Pat. No.4,594,810). The remaining complement is that of F118. The markers areinherited in codominant fashion and follow well-known rules of mendelianinheritance. That means if a hybrid has marker AB, and one of theparental lines has marker A (genotype AA), the other parent by inferencehas marker B. If the parent is an inbred line, it is expected to be BB,because it is essentially homozygous. The marker genetic complement ofDK743 is presented for RFLP and isozymes in Tables 3 and 4. The geneticcomplement of parental line HBA1, a patented line, may be determined byobtaining a sample of deposited seed, obtaining plant tissue, andfollowing the marker detection methods disclosed within. The parentalcontribution of F118 can be determined by inference. Because F118 andHBA1 are inbred lines, marker profiles are not disrupted by linkagedisequilibrium, and are likely to remain associated with adjacentgenetic segments.

Markers may also be used to identify and separate nucleic acid segmentsnecessary to produce the advantageous phenotype of DK743.

DEFINITIONS

Cross-pollination--pollen from a flower of one plant is used tofertilize a different maternal plant.

Elite Germplasm--in its area of adaptation, germplasm that is consideredby breeders to possess a high level of favorable traits.

Substantially Similar Genetic Complement--genetic complements havingnucleic acid sequences which code for the same exons or mutant orvariant sequences which either are codon equivalents or do not affectthe phenotype as disclosed herein.

Plant Components--all somatic cells, including protoplasts, calli, andparts of plants, from which plants can be regenerated in tissue culture,e.g., flowers, kernels, ears, cobs, leaves, husks, and stalks.

Self-pollination--pollen from one flower is transferred to the same oranother flower of the same plant.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects and advantages of the invention will become apparent uponreading the following detailed description and upon reference to thedrawings in which:

FIG. 1 is a relative maturity map used to define temperature ranges forcorn plant growth.

FIG. 2 is a graphic presentation of the reaction of DK743 to disease andinsects.

    ______________________________________                                        Code  Description                                                             ______________________________________                                        Foliar Diseases (Fungal)                                                      HM -  Helminthosporium maydis (Syn. = Bipolaris maydis)                             race 0, which causes southern corn leaf blight.                         HT1 & Helminthosporium turcicum (Syn. = Exserohilum                           HT2 - turcicum) races 1 and 2, which cause northern corn                            leaf blight.                                                            HC3 - Helminthosporium carbonum (Syn. = Bipolaris zeicola)                          race 3, which causes Helminthosporium leaf spot.                        CG -  Colletortrichum graminicola, which causes the foliar                          phase of anthracnose.                                                   KZ -  Kabatiella zeae, which causes eyespot of corn.                          CZ -  Cercospora zeae-maydis, which causes gray leaf spot                           of corn.                                                                CN -  Systematic Disease (Viral) Corynebacterium                                    nebraskense (Syn. = Clavibacter michiganense subsp.                           nebraskense), which causes Goss' disease.                               CLN - Corn Lethal Necrosis (combination of Maize Chlorotic                          Mottle Virus and Maize Dwarf Mosaic Virus).                             SVC - Southeastern Virus Complex (combination of Maize                              Chlorotic Dwarf Virus Insects and Maize Dwarf Mosaic                          Virus).                                                                 ON1 - Ostrinia nubilalis, first brood of European corn                              borer.                                                                  ON2 - Ostrinia nubilalis, second brood of European corn                             borer.                                                                  ______________________________________                                    

DESCRIPTION OF THE PREFERRED EMBODIMENT

This invention relates to a hybrid genetic complement produced bycombining the haploid genetic complements of two parental inbred linesof corn.

Corn has a diploid phase which means two conditions of a gene (twoalleles) occupy each locus (position on a chromosome). If the allelesare the same at a locus, there is said to be homozygosity. If they aredifferent, there is said to be heterozygosity. In a completely inbredline, all loci are homozygous. Because many loci when homozygous aredeleterious to the plant, in particular leading to reduced vigor,production of inbred lines is an unpredictable and arduous process.Under some conditions, heterozygous advantage at some loci retards theapproach toward homozygosity. Some recessive pairs are deleterious.

Inbreeding requires coddling and sophisticated manipulation by humanbreeders. Even in the extremely unlikely event inbreeding rather thancrossbreeding occurred in natural corn, achievement of completeinbreeding cannot be expected in nature due to well known deleteriouseffects of homozygosity and the large number of generations the plantwould have to breed in isolation. The reason for the breeder to createinbred lines is to have a known reservoir of genes whose gametictransmission is at least somewhat predictable.

The development of corn hybrids to generate crops with improvements incommercially desirable traits, is a sequential and laborious process ofrecurrent selection of inbred or partially inbred lines to form breedingpools from which new lines are further selected either byself-pollination and selection of desired phenotypes, and furtherinbreeding. Inbreds are then selected to be crossed with other inbredsto form hybrids which are evaluated in terms of their commercialpotential. Many generations of breeding are involved before finalselections are made of desirable hybrids for commercial use. "Uncommonlygreat genetic variability is present in and among the diverse lines,varieties, and races of corn in the world." (Coe et al., 1988, p. 111.)A breeders task is to delve into the morass of genetic variability andextract a new, improved combination of genes.

Herculean efforts have been undertaken, particularly in the 1900's bycommercial seed producers in continuous research and developmentdirected toward crop improvement. Selection and development of inbredlines is tenuous and tedious. Challenges posed by varying climates, newpests, and economic trends, prevent successful selection of "off theshelf" inbred lines to get a hybrid with desired traits. Troyer (1990),chronicles the excruciatingly tedious, complex venture of corn breedingfor crop improvement. Although there are some well-established cornbreeding techniques, they are not cook-book protocols which can beimplemented without significant human interaction. Moreover, they areguidelines for improvement, not blueprints for success.

Sprague and Eberhart (1977) illustrate the complexity of the involvementof the human breeder in corn programs. More importantly, Spraguediscusses the relevance of additive and non-additive genetic effects. Inadditive effects, the genes contributed to an offspring by a certainline produce characteristic effects, usually regardless of the order ofpairing, when combined with genetically analogous germplasm.Non-additive effects, however, must be considered in predictingphenotypic effects resulting from crossing certain genotypes. Dominance(intra-locus genetic interactions) and epistasis (inter-locus effects)are not completely predictable functions of the simple combinations ofparental traits.

It is not expected that a parental line with superior traits will conveythese traits to offspring of a cross with another parental line.Transmission of traits depends on the nature of the genetic transmissionand the basis for the phenotypic trait. Those traits due to recessiveinheritance in either parent, are expected to disappear or be modifiedin a hybrid because the homozygosity underlying the trait is disruptedunless identical alleles are donated from each parent to the relevantloci in the hybrid. The goal is that the disruption is beneficial, thatis, heterosis results. For any one cross that turns out to be superior,many, many such crosses will likely fail. Interactions among loci(epistasis) are also generally unpredictable in offspring of new geneticparental combinations.

The performance of an inbred line is judged by its performance whencrossed with other inbred lines to produce hybrids.

The general combininq ability of a particular inbred is determined byits average performance in a series of hybrid combinations. Generalcombining ability is said to measure mainly additive genetic effects.Specific combininq ability (sca) refers to the performance of twoparticular inbreds in a specific cross. Specific combining abilitymeasures nonadditive types of gene interactions.

One of the traits for which hybridization has been employed to causeimprovement is yield per acre of corn. For example, in the Apr. 17, 1974edition of the Wall Street Journal, an article entitled "In Search ofSuperbean," it was pointed out that soybeans could not easily behybridized and therefore fell far behind corn and productivityincreased. During the period of 1950 to 1973, soybeans increased inproductivity from 21.8 to 27.8 bushels per acre, whereas corn increasedfrom 38.4 to 91.4 bushels per acre. Improvements since that time havebeen sought by sophisticated plant and genetic manipulation.

Whether two particular inbreds combine to produce a high-yielding singlecross depends upon the extent to which favorable genes for yield fromone inbred supplement those contributed by the second inbred.

Mixing parental germplasm from inbred lines in the cauldron of the F₁hybrid does not lead to a completely predictable spectrum of traits inthat hybrid. This is because traits in the hybrid result frominteractions among genes at the same locus (dominance, overdominance),genes at different loci (epistasis) and genes and environments. Themeiotic shuffling of the genes plus the interaction of genes with theenvironment will lead to the trait. It is the trait which is sought ascommercially desirable.

Hybrids may be produced from single or multiple parental inbred linecrosses, although of course only two parents may be used to produce anyparticular plant. However, successive single cross hybrids fromdifferent inbred lines will lead to the merging of the genetic parentalcomplements of various inbred lines into one hybrid. It is this matrixof multiple merging that illustrates the sophisticated methods ofselection and crop improvement.

Complex mathematical models have been developed to predict offspringphenotypes based on parental genotypes and the environment (Falconer,1960; Sprague and Eberhart, 1977).

Breedinq Methods to Develop the Hybrid Genetic Complement of the PresentInvention

The inbred lines used in this invention were HBA1, a patented inbredline (U.S. Pat. No. 4,594,810) and F118 a proprietary line. They werecrossed to produce the hybrid DK743.

Based on well-established genetic principles, it is known that ahomozygous line crossed to another homozygous line is expected to yielda hybrid, usually designated F₁, whose plants are genotypically the samebarring rare events such as mutation. Of course, the phenotype of thehybrid organism may not always be identical when the same inbred linesare used as parents because the comparability of the phenotypicexpression of the genotype depends on the similarity of theenvironmental conditions interacting with the genotype to produce thephenotype. In general, the phenotypes are expected to be similar unlessthe phenotype or aspects thereof is strongly dependent on environmentalconditions.

Reciprocal crosses refer to interchange of the sex of the parents. Formany crosses, the outcome is the same regardless of the assigned sex ofthe parental lines. However, there is often one of the parental linesthat is preferred as the maternal line because of increased seed yieldand production characteristics. Some lines produce tighter ear husksleading to more loss, for example due to rot. There may be delays insilk formation which deleteriously affect timing of the reproductivecycle for a pair of parental inbreds. Seed coat characteristics may bepreferable in one line. Pollen may be shed better by one line. Othervariables may also affect preferred sexual assignment of a particularcross.

Because there is no adequately reliable way to predict the overallphenotype of any particular F₁ hybrid, tens of thousands of hybridsresulting from large numbers of crosses are required and must beevaluated each year. These hybrids are then screened and evaluated insmall scale field trials. ("R" or research trials, Table 2). Typically,about 10-15 phenotypic traits are measured. These traits are selectedfor their potential commercial value. A selection index of the mostcommercially important traits is used to help evaluate hybrids. ForDK743 yield, moisture, percent dropped, and percent lodged were used inthe selection index.

After one year of evaluation, approximately 95% of the hybrids arerejected on the basis of their performance not being above that ofpreviously developed hybrids.

During the next several years, a progressive elimination of hybridsoccurs based on more detailed evaluation of their phenotype. Eventually,strip trials (FACT) are conducted to formally compare the experimentalhybrids being developed with other hybrids, some of which werepreviously developed and generally are commercially successful. FACT, anacronym for Field Analysis Comparison Trial (strip trials), is anon-farm testing program employed by DeKalb Plant Genetics to perform thefinal evaluation of the commercial potential of a product. (See Table 1for DK743 results as compared with some competitors.) That is,comparisons of the experimental hybrids were made to competitive hybridsin order to determine if there was any advantage to further commercialdevelopment of the experimental hybrids. Comparisons were made tohybrids of similar or identical maturity.

Time to maturity is an important characteristic for a corn line and mustmatch the environment in which the corn is to be planted. If there isonly enough heat accumulated to mature a hybrid corn plant in 80 days,and the plant requires 125 days, there will not be time enough to entera reproductive phase. Maturity of a hybrid is not completely predictablefrom that of the parental inbreds. Another complication is that therange of maturity will be based on the selection environments.

Strip trials compare the phenotypes of hybrids grown in as manyenvironments as possible. Strip tests were performed in manyenvironments to assess overall performance of the new hybrids and toselect optimum growing conditions. The corn being compared is of similarmaturity. The corn is grown in close proximity at each location,consequently, environmental factors that affect gene expression areminimized for that location. These factors include moisture,temperature, diseases, sunlight and pests.

For a decision to be made that a hybrid is worth making commerciallyavailable, it is not necessary that the hybrid be better than all otherhybrids. Rather, significant improvements must be shown in at least sometraits that would create improvements in some niches, in particular inwhat is considered to be the target market.

When the two inbred parental lines F118 and HBA1 were crossed to producethe hybrid DK743, either parent may serve as the maternal or paternalplant. However, in the production of DK743, F118 is preferred as thefemale, HBA1 as the male, parent, because of better quality seedproduction.

The hybrid genetic complement resulting from combining F118 with HBA1(designated DK743) has produced an unexpectedly high-yielding hybridline also with other desirable traits.

Some of the other desirable traits exhibited by DK743 are also importantcommercially, e.g., excellent staygreen, high silage production. Diseasetolerance is also excellent. (see Appendix for definitions of thesetraits). Tables 2-4 list identifying features and characteristics ofthese hybrids. Table 1 compares their performance with othercommercially successful hybrids. It is unexpected to improve severaltraits in the same hybrid. "Gains may be made in the defensive traitsbut not in yield, or new hybrids may be improved in yield but not indefensive traits." (Duvick, 1984, p. 47).

Results of experiments designed to test response of DK743 to insects andresistance to disease, are shown in FIG. 3. The levels of disease andinsect resistance make DK743 a highly acceptable hybrid in itsmarketplace.

Table 1 presents examples of comparisons of performance data for DK743versus selected hybrids of commercial value. These data representresults across years and locations for strip trials. The importantcomparisons are for competitors within the same geographical area inwhich the hybrid shows its best performance for traits of commercialvalue, e.g. yield.

The "NTEST" represents the number of paired observations in designatedtests at locations around the United States.

As can be seen in Table 1, DK743 has significantly higher yield whencompared to two commercially successful hybrids designated DK689 andPION3165. The increased yield in bushels per acre is 3.8 to 7.6.Significant improvements are also shown in Table 1 for many othertraits, notably staygreen. Staygreen is a scale 1-9 (9=best) of generalhealth of the plant after the ear leaves have reached maturity.

                                      TABLE 1                                     __________________________________________________________________________    COMPARISON OF DK743 WITH 2 COMMERCIALLY SUCCESSFUL HYBRIDS                    NTEST      SI %                                                                             YIELD MOIST                                                                              SV FGDU                                                                              PHT EHT                                                                              BAR                                                                              SG D % STL                                                                              RTL                                                                              TNT                                                                              ESTR                __________________________________________________________________________    DK743 R 88 110.5                                                                            158.9 21.0 6.0                                                                              1321                                                                              104.2                                                                             47.6                                                                             0.0                                                                              6.5                                                                              0.0 2.7                                                                              3.1                                                                              55.5                                                                             123.7               DK689      101.6                                                                            147.6 19.3 6.9                                                                              1302                                                                               99.4                                                                             50.5                                                                             0.0                                                                              5.2                                                                              0.2 4.9                                                                              1.9                                                                              54.7                                                                             120.4                          ** **    **   *  *   **  **    ** *   ** +                         DK743 F 176                                                                              103.0                                                                            125.9 19.1                     0.0 4.5                                                                              2.4                                                                              56.2                                                                             122.8               DK689       99.9                                                                            118.3 17.7                     0.3 3.7                                                                              0.5                                                                              56.5                                                                             118.6                          *  **    **                       **     *  **                     DK743 R 110                                                                              111.6                                                                            157.2 21.0 6.4                                                                              1320                                                                              103.3                                                                             47.9                                                                             0.0                                                                              6.4                                                                              0.0 2.6                                                                              3.8                                                                              55.5                                                                             124.1               PION3165    99.3                                                                            147.1 21.3 6.2                                                                              1370                                                                               98.7                                                                             48.5                                                                             0.0                                                                              6.7                                                                              0.2 3.7                                                                              4.5                                                                              55.2                                                                             124.6                          ** **    **      **  **           *   *                            DK743 F 107                                                                              104.4                                                                            121.4 18.3                     0.0 3.7                                                                              1.6                                                                              56.3                                                                             123.1               PION3165    99.6                                                                            117.6 18.5                     0.2 4.4                                                                              1.5                                                                              56.9                                                                             123.8                          ** *     *                        +         **                     __________________________________________________________________________     Where                                                                         R = research test results                                                     F = FACT testing                                                              SI % = Selection index, see 67 in appendix.                                   Yield = see 68 in appendix.                                                   Moist = see 69 in appendix.                                                   SV = seedling vigor, a 1-9 rating of early season plant growth where 9 =      best.                                                                         FGDU = energy required to flower (see 66 in this appendix).                   PHT = see 6 in appendix.                                                      EHT = see 41 in appendix.                                                     BAR = % barren, % of plants that lack an ear.                                 SG = see 70 in appendix.                                                      D % = see 71 in appendix.                                                     STL = % of plants broken over below the ear at harvest.                       RTL = % of plants leaning at greater than 30° angle to the ground.     TWT = see 65 in appendix.                                                     ESTR = estimate of relative maturity (days)                                   and significance levels are indicated as                                      + = 10%                                                                       * = 5%                                                                        ** = 1%     A general description of the DK743 hybrid is presented in         Table 2.

                  TABLE 2                                                         ______________________________________                                        MORPHOLOGICAL CHARACTERISTICS                                                 OF THE DK743 PHENOTYPE                                                        (See Appendix)                                                                CHARACTERISTIC        DK 743 VALUE*                                           ______________________________________                                        1.    Seedling                                                                      Color               Dark Green                                                Vigor 1-5 Rating    4                                                   2.    Stalk                                                                         Plant Height cm.    296.0                                                     Ear Height cm.      118.9                                                     Diameter (Width) cm.                                                                              2.5                                                       Anthocyanin         Basal strong                                              Nodes With Brace Roots                                                                            1.3                                                       Brace Root Color    Red                                                       Internode Direction Straight                                                  Internode Length cm.                                                                              17.6                                                3.    Leaf                                                                          Angle               Upright                                                   Number              20.8                                                      Post Poll Color     Dark Green                                                Length cm.          94.3                                                      Width cm.           9.5                                                       Sheath Anthocyanin  Weak                                                      Sheath Pubescence   Medium                                                    Marginal Waves      Many                                                      Longitudinal Creases                                                                              Absent                                              4.    Tassel                                                                        Total Length cm.    39.3                                                      Spike Length cm.    30.2                                                      Peduncle Length cm. 13.6                                                      Attitude            Compact                                                   Branch Angle        Intermediate                                              Branch Number       9.4                                                       Anther Color        Green Yellow                                              Glume Color         Green                                                     Glume Band          Absent                                              5.    Ear                                                                           Silk Color          Pink                                                      Number Per Stalk    1.1                                                       Position (Attitude) Upright                                                   Length cm.          16.7                                                      Shape               Semi-Conical                                              Diamter cm.         4.4                                                       Weight gm.          182.4                                                     Shank Length cm.    9.2                                                       Shank Internode Number                                                                            6.7                                                       Husk Bract          Short                                                     Husk Cover cm.      2.9                                                       Husk Opening        Intermediate                                              Husk Color Fresh    Green                                                     Husk Color Dry      Buff                                                      Cob Diameter cm.    2.4                                                       Cob Color           Red                                                       Cob Strength        Weak                                                      Shelling Percent    87.9                                                6.    Kernel                                                                        Row Number          15.6                                                      Number Per Row      38.1                                                      Row Direction       Curved                                                    Type                Dent                                                      Cap Color           Yellow                                                    Side Color          Orange                                                    Length (Depth) mm.  12.1                                                      Width mm.           8.1                                                       Thickness           3.6                                                       Weight of 1000K gm. 308                                                       Endosperm Type      Normal                                                    Endosperm Color     Yellow                                              7.    Other                                                                         Uniformity 1-5 Rating                                                                             1                                                         GDUS to 50% Pollen Shed                                                                           1460                                                      GDUS to 50% Silking 1436                                                ______________________________________                                         *These are typical values of DK743 plants. Values may vary due to             environment. Other values that are substantially equivalent are also          within the scope of the invention. "Substantially equivalent" refers to       quantitative traits that when compared do not show statistically              significant differences of their means.                                  

Genetic Markers to Identify Plants

Markers are genes, the phenotypic expressions of which are used toidentify the presence of other genes or genetic complements whichcosegregate with the markers through meiosis and appear jointly inoffspring. Markers are generally codominant, that is, both alleles at amarker locus are readily detectable in a heterozygote. Markers which areuseful in plant breeding comprise isozymes and restriction fragmentlength polymorphism (RFLP's). RFLP analysis has been performed on theparents of DK743 leading to the hybrid profile shown in Table 3.

Isozymes are forms of proteins that are distinguishable, for example, onstarch gel electrophoresis, usually by charge and/or molecular weight.The isozyme profile of DK743 is shown in Table 4.

A standard set of loci may be used as a reference set. Comparativeanalysis of these loci may be used to compare the purity of hybridseeds, to assess the increased variability in hybrids compared toinbreds, and to determine the identity of seeds, plants, and plantparts. In this respect, an isozyme or RFLP reference set may bepartially used to develop genotypic "fingerprints." (See Evola et al.;Helentjaris et al., 1985, 1986).

Table 4 lists the identifying numbers of the alleles segregating atisozyme loci types for hybrid DK743. This set of alleles are one set ofidentifiers for the genetic complements of this hybrid. These allelesare not known to be directly related to the desirable traits of thesehybrid plants (Goodman and Stuber, 1980), but are useful to identifygenomes.

                  TABLE 3                                                         ______________________________________                                        ALLELES IN DK743 DETECTED BY RESTRICTION                                      ENDONUCLEASE DIGESTION OF GENOMIC                                             DNA FOLLOWED BY TREATMENT WITH PROBES                                         Probe/Enzyme Combination                                                                             Allelic Pair                                           ______________________________________                                        0264H                  EG                                                     0285E                  DE                                                     0306H                  AA                                                     0445E                  BD                                                     0B304E                 --                                                     1120S                  BB                                                     1234H                  AE                                                     1236H                  --                                                     1238H                  AA                                                     1401E                  BC                                                     1406H                  AB                                                     1447E                  --                                                     1447H                  AD                                                     1B725E                 BH                                                     2239H                  --                                                     2297H                  AE                                                     2298E                  DF                                                     2402H                  EE                                                     3212S                  --                                                     3247E                  --                                                     3257S                  --                                                     3296H                  AA                                                     3432H                  FH                                                     3446S                  --                                                     3457E                  EF                                                     3B815H                 BB                                                     4386H                  AD                                                     4396E                  --                                                     4444H                  ADG                                                    4451H                  BG                                                     4UMC19H                AC                                                     4UMC31E                --                                                     4UMC31S                --                                                     5213S                  AB                                                     5288E                  BC                                                     5288S                  --                                                     5295E                  DC                                                     5408H                  AA                                                     5409H                  CD                                                     5579S                  --                                                     5UMC95H                CD                                                     6223E                  BC                                                     6252H                  AE                                                     6280H                  AB                                                     6373E                  AE                                                     7263E                  AB                                                     7391H                  AA                                                     7392S                  --                                                     7433E                  AD                                                     7455H                  BC                                                     8107S                  CD                                                     8110S                  CD                                                     8114E                  EG                                                     8268H                  AB                                                     8438E                  CC                                                     8585H                  --                                                     8B2369S                --                                                     8UMC48E                CC                                                     9209E                  AA                                                     9211E                  --                                                     9266S                  --                                                     9B713S                 AB                                                     9BZE                   AA                                                     9WAXE                  GG                                                     ______________________________________                                         *Probes used to detect RFLP's are from Native Plants Incorporated, 417        Wakara Way, Salt Lake City, Utah, 84108. See EPO 306 139 A2; probes are       currently available from Linkage Genetics, 1515 W. 2200 S., Suite C, Salt     Lake City, Utah 84119.                                                   

                  TABLE 4                                                         ______________________________________                                        ISOZYME GENOTYPE FOR HYBRID DK743                                             LOCUS             ISOZYME ALLELES                                             ______________________________________                                        Acph              2                                                           Adh1              4                                                           Cat3              9/12                                                        Dia-1             8                                                           Dia-2             4                                                           Got-3             4                                                           Got-2             4                                                           Got-1             4                                                           Idh-1             4                                                           Idh-2             4/6                                                         Mdh-1             6*                                                          Mdh-2             3.5/6                                                       Mdh-3             16                                                          Mdh-4             12                                                          Mdh-5             12                                                          Pgm-1             9                                                           Pgm-2             4                                                           6-Pgd-1           3.8                                                         6-Pgd-2           5                                                           Phi-1             5                                                           Tpi-1             4                                                           Tpi-2             4                                                           Tpi-3             4                                                           Tpi-4             4                                                           # of Seeds Assayed                                                                              6                                                           ______________________________________                                         *Allele is probably a 6, but null cannot be ruled out.                   

Methods for In Vitro Hybrid Plant Regeneration

Hybrid plants can be grown from hybrid seeds by methods well known tothose skilled in the art. Hybrid plants may also be regenerated fromtissues of hybrid plants by use of in vitro laboratory methods of tissueculture.

In certain embodiments, recipient cells are selected following growth inculture. Where employed, cultured cells are preferably grown either onsolid supports or in the form of liquid suspensions. In either instance,nutrients are provided to the cells in the form of media, andenvironmental conditions are controlled. There are many types of tissueculture media comprising amino acids, salts, sugars, hormones andvitamins. Most of the media employed to regenerate hybrid plants willhave some similar components, the media differ in the composition andproportions of their ingredients depending on the particular applicationenvisioned. For example, various cell types usually grow in more thanone type of media, but will exhibit different growth rates and differentmorphologies, depending on the growth media. In some media, cellssurvive but do not divide.

Various types of media suitable for culture of plant cells have beenpreviously described. Examples of these media include, but are notlimited to the N6 medium described by Chu, et al. (1975) and the MSmedia, Murashige and Skoog (1962). In an exemplary embodiment forpreparation of recipient cells, modifications of these media areavailable. A preferred hormone for such purposes is dicamba or 2,4-D.However, other hormones may be employed, including NAA, NAA+ 2,4-D orperhaps even picloram. Modifications of these and other basic media mayfacilitate growth of recipient cells at specific developmental stages.

An exemplary embodiment for culturing recipient corn cells in suspensioncultures includes using embryogenic cells in Type II (Armstrong andGreen, 1985; Gordon-Kamm, et al., 1990) callus, selecting for small(10-30μ) isodiametric, cytoplasmically dense cells, growing the cells insuspension cultures with hormone containing media, subculturing into aprogression of media to facilitate development of shoots and roots, andfinally, hardening the plant and readying it metabolically for growth insoil.

Meristematic cells (i.e., plant cells capable of continual cell divisionand characterized by an undifferentiated cytological appearance,normally found at growing points or tissues in plants such as root tips,stem apices, lateral buds, etc.) may be cultured.

EXAMPLES Example 1: Methods of Producing Hybrid Genetic Complements:Breeding

Corn plants (Zea mays L.) can be bred by either self-pollination orcross-pollination techniques. Corn has male flowers, located on thetassel, and female flowers, located on the ear, on the same plant.Natural pollination occurs in corn when wind blows pollen from thetassels to the silks that protrude from the tops of the incipient ears.Mechanical pollination may be effected either by controlling the typesof pollen that can blow onto the silks or by pollinating by hand.

Two inbred lines were selected as parents, one of which is the inbredline F118, the other HBA1. HBA1 is a patented inbred line (U.S. Pat. No.4,594,810). F118 is a proprietary line. These parental lines wereplanted in pollinating proximity to each other. This may be achieved byplanting the parental lines in alternating rows, in blocks or in anyother convenient planting pattern. The plants of both lines were allowedto grow until the time of flowering. Advantageously, during this growthstage, the plants are in general treated with fertilizer and/or otheragricultural chemicals as considered appropriate by the grower.

At the time of flowering, in the event that inbred line HBA1 is employedas the male parent, the tassels of the other parental line, F118 areremoved from all plants employed as the female parental line. Thedetasseling can be achieved manually but also can be done by machine ifdesired.

The lines are then allowed to continue to grow and naturalcross-pollination occurs as a result of the action of wind which isnormal in the pollination of grasses, including corn. As a result of theemasculation of the female parent line, all the pollen from the maleparent line, e.g., HBA1, is available for pollination because tassels,and thereby pollen bearing flowering parts, have been previously removedfrom all plants of the inbred line being used as the female in thehybridization. Of course, during this hybridization procedure, theparental varieties are grown such that they are isolated from other cornfields to prevent any accidental contamination of pollen from foreignsources. These isolation techniques are well within the skill of thoseskilled in this art.

Both of the parent inbred lines of corn are allowed to continue to growuntil maturity, but only the ears from the female inbred parental linesare harvested to obtain seeds of a novel F_(l) hybrid corn. If desired,corn produced in the male parent variety can be harvested, e.g., forgrain use, but these seeds are not useful as hybrid seeds.

The novel F₁ hybrid seed produced may then be planted in a subsequentgrowing season with the desirable characteristics in terms of F₁ hybridcorn plants providing improved grain yields and the other desirablecharacteristics disclosed herein, being achieved.

Example 2: Methods of Producing Hybrid Genetic Complements In VitroRegeneration

Embryogenic calli are produced (Gordon-Kamm et al., 1990). Specifically,plants from hybrids produced from crossing the inbreds (F118×HBA1) aregrown to flowering in a greenhouse. Explants from at least one of thefollowing F₁ tissues: immature tassel tissue, intercalary meristems andleaf bases, apical meristems, and immature ears are placed in aninitiation medium which contain MS salts, supplemented with thiamine,agar, and sucrose. Cultures are incubated in the dark at about 23° C.All culture manipulations and selections are performed with the aid of adissecting microscope.

After about 5-7 days, cellular outgrowths are observed from the surfaceof the explants. After about 7-21 days, the outgrowths are subculturedby placing into fresh medium of the same composition. Some of the intactexplants are placed on fresh medium.

Several subcultures later (after about 2 to 3 months) enough material ispresent from explants for subdivision of these embryogenic calli intotwo or more pieces. Callus pieces from different explants are not mixed.After further growth and subculture (about 6 months after embryogeniccallus initiation), there are usually between 1 and 100 pieces derivedultimately from each selected explant. During this time of cultureexpansion, a characteristic embryogenic culture morphology develops as aresult of careful selection at each subculture. Any organized structuresresembling roots or root primordia are discarded. Any material knownfrom experience to lack the capacity for sustained growth is alsodiscarded (translucent, watery, embryogenic structures). Structures witha firm consistency resembling at least in part the scutulum of the invivo embryo are selected.

The callus is maintained on agar-solidified MS-type media. The hormoneused is 2,4-D. Visual selection of embryo-like structures is done toobtain subcultures. Transfer of material other than that displayingembryogenic morphology results in loss of the ability to recover wholeplants from the callus.

Some calli will exhibit somaclonal variation. These are genotypicchanges appearing in culture.

Cell suspensions are prepared from the calli by selecting cellpopulations that appear homogeneous macroscopically. A portion of thefriable, rapidly growing embryogenic calli is inoculated into MS Medium.The calli in medium are incubated at about 27° C. on a gyrotary shakerin the dark or in the presence of low light. The resultant suspensionculture is transferred about once every seven days by taking about 5 to10 ml of the culture and introducing this inoculum into fresh medium ofthe composition listed above.

For regeneration, embryos which appear on the callus surface areselected and regenerated into whole plants by transferring theembryogenic structures into a sequence of solidified media which includedecreasing concentrations of 2,4-D or other auxins. Other hormones whichmay be used in the media include dicamba, NAA, ABA, BAP, and 2-NCA. Thereduction is relative to the concentration used in culture maintenancemedia. Plantlets are regenerated from these embryos by transfer to ahormone-free medium, subsequently transferred to soil, and grown tomaturity.

Progeny are produced by taking pollen and selfing, backcrossing orsibbing regenerated plants by methods well known to those skilled in thearts. Seeds are collected from the regenerated plants.

While the invention is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof examples and described herein in detail. It should be understood,however, that it is not intended to limit the invention to theparticular forms disclosed, but on the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the appended claims.

APPENDIX PLANT TRAIT DESCRIPTIONS

2. Seedling Emergence--Number of emerged plants divided by number ofseeds planted, expressed as a percentage.

3. Seedling Height--Plant height at the 6-8 leaf stage in centimeters.

4. Seedling Color--Color of leaves at the 6-8 leaf stage (light green,dark green, anthocyanin).

5. Seedling Vigor--Rated on a scale of 1-5 (1=best) at the 2-4 leafstage.

6. Plant Height--Measured from soil to the tip of the tassel incentimeters.

7. Stalk Diameter--Diameter of the lowest visible internode, measured incentimeters.

8. Stalk Anthrocyanin--Rated 1-2 weeks after pollination as absent,basal-weak, basal-strong, weak, strong.

9. Stalk Nodes with Brace Roots--Average number of nodes having braceroots on each plant.

10. Stalk Brace Root Color--Color of brace roots observed 1-2 weeksafter pollination (green, red, or purple).

11. Stalk Internode Length--Length of the internode above the primaryear in centimeters.

12. Stalk Internode Direction--Observed after pollination, straight orzig-zag.

13. Leaf Angle--Angle of the upper leaves to the stalk; 0°-30° =upright,30°-60° =intermediate, 60°-90° =lax.

14. Leaf Number--Total number of leaves from the cotyledon leaf to theflag leaf.

15. Leaf Length--Length of the primary ear leaf measured in centimeters.

16. Leaf Width--Width of the primary ear leaf measured at the widestpoint in centimeters.

17. Leaf Color--Color of the leaves 1-2 weeks after pollination. (Lightgreen, medium green, dark green, very dark green).

18. Leaf Marginal Waves--Rated for waves on the leaf margin 1-2 weeksafter pollination (none, few, many).

19. Leaf Longitudinal Creases--Rated for creases on the leaf surface 1-2weeks after pollination (absent, few or many).

20. Leaf Sheath Anthocyanin--Rated 1-2 weeks after pollination (absent,basal-weak, basal strong, weak, strong).

21. Leaf Sheath Pubescence--Rated 1-2 weeks after pollination (light,medium or heavy).

22. Tassel Length--Measured from the base of the bottom tassel branch tothe tassel tip in centimeters.

23. Tassel Spike Length--Measured from the base of the top tassel branchto the tassel tip in centimeters.

24. Peduncle Length--Measured from the base of the flag leaf to the baseof the bottom tassel branch in centimeters.

25. Tassel Branch Number--Number of primary tassel branches.

26. Tassel Branch Angle--Angle of an average tassel branch to the mainstem of the tassel; less than 30° =upright, 30°-45° =intermediate,greater than 45° =lax.

27. Tassel Attitude--Observed after pollination as open or compact.

28. Tassel Glume Color--Color of glumes at 50% shed (green, red orpurple).

29. Tassel Glume Band--Closed anthocyanin ring at base of glume (absentor present).

30. Tassel Anther Color--Color of anthers at 50% shed (green-yellow,yellow, pink, red, purple).

31. 50% Shed Standard GDUS--Standardized GDUS are adjusted for thelocation and year affects (1989 DeKalb factor=-115. 1989 Illiopolisfactor =+24).

32. 50% Silk Standard GDUS--Standardized GDUS are adjusted for thelocation and year affects (1989 DeKalb factor =-115, 1989 Illiopolisfactor=+24).

33. Uniformity--Rated throughout the growing season based on variabilityin plant & ear height, tassel type, anther and silk color (scale of 1-5;1=best).

34. Husk Bract--Length of an average husk leaf (short, medium or long).

35. Husk Cover--Distance from the tip of the ear to the tip of the husks(exposed ear=0.0).

36. Husk Opening--Husks are rated for tightness at harvest stage (tight,intermediate or open).

37. Husk Color Fresh--Color of husks 1-2 weeks after pollination (green,red or purple).

38. Husk Color Dry--Color of husks at harvest stage (buff, red orpurple).

39. Shank Length--Length of the ear shank measured in centimeters.

40. Shank Internode Number--Number of internodes on the ear shank.

41. Ear Height--Measured from the soil to the base of the primary ear incentimeters.

42. Ears/Stalk--The number of ears with grain on each plant.

43. Ear Silk Color--Color of silk observed 2-3 days after silks emerge(green-yellow, yellow, pink, red, purple).

44. Ear Position--Rated at harvest stage (upright, horizontal, pendent).

45. Ear Weight--Weight of the ear expressed in grams.

46. Ear Length--Length of the ear measured in centimeters.

47. Ear Diameter--Diameter of the ear at the midpoint measured incentimeters.

48. Ear Shape--Observed as conical, semi-conical or cylindrical.

49. Kernel Row Number--Number of rows on one ear.

50. Kernel Row Direction--Observed as straight, slightly curved, spiralor indistinct (scattered).

51. Kernel Number per Row--Number of kernels in one row.

52. Kernel Cap Color--Color of the kernel cap observed at dry stage(white, lemon-yellow, yellow, orange).

53. Kernel Side Color--Color of the kernel side observed at dry stage(white, pale yellow, yellow, orange, red, brown).

54. Kernel Endosperm Type--Observed as normal, waxy, opaque.

55. Kernel Endosperm Color--Observed as white, pale yellow, yellow.

56. Kernel Weight/1000k--Weight of 1000 kernels expressed in grams.

57. Kernel Length--Distance from the cap to the pedicel measured inmillimeters.

58. Kernel Width--Distance across the flat side of the kernel measuredin millimeters.

59. Kernel Thickness--Distance across the narrow side of the kernelmeasured in millimeters.

60. Kernel Type--Observed as dent, flint, intermediate.

61. Cob Diameter--Diameter of the cob at the midpoint measured incentimeters.

62. Cob Strength--Cobs are mechanically broken and rated as eitherstrong or weak.

63. Cob Color--Color of the cob (white, pink, red, brown, purple).

64. Shelling Percentage--Shelled grain weight divided by the sum ofgrain and cob weight, expressed as a percentage.

65. Test Weight--U.S. Government standards are that 56 lbs equals 1bushel, with a 15% moisture content.

66. GDU Shed--The number of growing degree units (GDU) required for aninbred line or hybrid to shed pollen from the time of planting. ##EQU1##Where maximum daily temperature must not exceed 85° F. and minimum mustnot be below 50° F.

67. Selection Index--A single measure of a hybrid's relative worth basedon information for up to five traits (always includes yield).

68. Yield--Actual bushels/acre at harvest adjusted to 15.5% moisture.

69. Moisture--Per cent moisture of the grain at harvest.

70. Staygreen--A measure of plant health at the time of maturity of theear leaves.

71. Dropped Ears--The percent of plants that did not drop ears prior toharvest.

REFERENCES

The references listed below are incorporated herein by reference to theextent that they supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

Reference 1. Armstrong and Green, (1985). "Establishment and Maintenanceof Friable, Embryogenic Maize Callus and the Involvement of L-Proline,"Planta, 164:207-214.

Reference 2. Asins, M. J. (1988) "Detection of Linkage BetweenRestriction Fragment Length Polymorphism Markers and QuantitativeTraits," Theor. App. Genet. 76:623-626.

Reference 3. Beckmann, J. S. and Soller, M. (1983) "Restriction FragmentLength Polymorphisms in Genetic Improvement: Methodologies, Mapping andCosts," Theor. Appl. Genet. 67:35-43.

Reference 4. Burr, B., Evola, E., Burr, F., et al. (1983) "TheApplication of Restriction Fragment Length Polymorphisms to PlantBreeding," Settoco, J. K. and Hollaender, A. (eds.) Genetic EngineeringPrinciples and Methods, Plenum Press, N.Y. and London, pp. 45-59.

Reference 5. Chu, C. C., Wang, C. C., Sun, C. S., et al. (1975).Scientia Sinica 18:659-668.

Reference 6. Coe, E. H., et al (1988) "The Genetics of Corn and CornImprovement", 3rd. ed., Vol. 18, Sprague and Dudley (eds) 87:258

Reference 7. Duvick, D. N. (1984) "Genetic Contribution to Yield Gainsof U.S. Hybrid Maize--1930-1980," Genetic Contribution to Yield Gains ofFive Major Crops, pp. 15-48.

Reference 8. Evola, S. V., Burr, J. A., and Burr, B. (1986) "TheSuitability of Restriction Fragment Length Polymorphisms as GeneticMarkers in Maize." Theor. App. Genet. 71:765-771.

Reference 9. Falconer, D.S. (1960) "Introduction to QuantitativeGenetics," Ronald Press Co., New York.

Reference 10. Goodman, M. and Stuber, C. (1980), "Genetic Identificationof Lines and Crosses Using Isoenzyme Electrophoresis," Proceedings ofthe Thirty-Fifth Annual Corn and Sorghum Industry Research Conferences,Chicago, 1980.

Reference 11. Gordon-Kamm, W. et al., (1990) "Transformation of MaizeCells and Regeneration of Fertile Transgenic Plants," The Plant Cell, V.2, 603-618.

Reference 12. Helentjaris, T., King, G., Slocum, M., et al. (1985)"Restriction Fragment Polymorphisms as Probes for Plant Diversity andTheir Developments as Tools for Applied Plant Breeding," Plant Mol.Biol. 5:109-118.

Reference 13. Helentjaris, T., Slocum, M. Wright, S., et al., (1986)"Construction of Genetic Linkage Maps in Plants Using RestrictionFragment Polymorphisms," Theor. Appl. Genet. 72:761-769.

Reference 14. Lande, R. and Thompson, R. (1990) "Efficiency ofMarker-Associated Selection in the Improvement of Quantitative Traits,"Genetics 124:743-756.

Reference 15. Lander, E.S. and Botstein, D. (1989) "Mapping MendelianFactors Underlying Quantitative Traits Using RFLP Linkage Maps,"Genetics 121:185-199, see also WO 90104651.

Reference 16. Murashige, T. and Skoog, F. (1962). Plant Physiol.15:473-497.

Reference 17. Paterson, A. H., et al. (1988) "Resolution of QuantitativeTraits into Mendelian Factors by Using a Complete Linkage Map ofRestriction Fragment Polymorphism," Nature 335:721-724 .

Reference 18. Rhodes, C. A., Pierce, D. A., Mettler, F. J., et al.(1988). Science 240:204-207.

Reference 19. Sprague, G. F. and Eberhart, S. A. (1977) "Corn Breeding,"in Corn and Corn Improvements, J. A. Dudley and G. F. Sprague (eds),Iowa State Univ. Press.

Reference 20. Troyer, A. F. (1990) "A Retrospective View of Corn GeneticResources", Journal of Heredity, 81:17-24.

Reference 21. EP 0 306 139 A2, "Identification, Localization andIntrogression into Plants of Desired Multigenic Traits."

Reference 22 PCT/US89/00709, "Genetic Linkages Between AgronomicallyImportant Genes and Restriction Fragment Length Polymorphisms."

What is claimed is:
 1. A hybrid corn plant designated DK743 formed bythe crossing of inbred corn plants HBA 1, having ATCC No. 40225 and F118having ATCC accession No.
 75745. 2. A seed of the corn plant accordingto claim
 1. 3. A hybrid corn plant having all phenotypic andmorphological characteristics of the corn plant according to claim
 1. 4.A tissue culture of regenerable cells of DK743.